CN117599157A - Application of NIH3T3 mouse fibroblast autophagosome in preparation of vaccine for treating melanoma - Google Patents

Abstract

The invention discloses an application of an NIH3T3 mouse fibroblast autophagy body in preparing a vaccine for treating melanoma, wherein the NIH3T3 mouse fibroblast autophagy body is prepared from NIH3T3 mouse fibroblasts which stably express a new melanoma antigen and MAP1LC 3. According to the invention, the mouse fibroblast NIH3T3 autophagosome is modified through genetic engineering, so that the mouse fibroblast NIH3T3 autophagosome is coated with a new melanoma antigen and MAP1LC3 at the same time, and the immune system can be activated, thereby achieving the purpose of inhibiting the growth of melanoma; in addition, the method adopts two medicaments to jointly treat NIH3T3 cells, and can rapidly extract a large amount of autophagosomes by a grinding and centrifuging method.

Description

Application of NIH3T3 mouse fibroblast autophagosome in preparation of vaccine for treating melanoma

Technical Field

The invention relates to the technical field of vaccines, in particular to application of an NIH3T3 mouse fibroblast autophagosome in preparation of a vaccine for treating melanoma.

Background

Melanoma is a malignant tumor derived from melanocytes, and is the tumor with the highest malignancy among skin cancers. Once melanoma is metastasized, the survival rate of patients in 5 years is only 4.6%, and the melanoma has high mortality, high metastasis rate and high treatment difficulty. Thus, there is an urgent clinical need to find effective treatments for melanoma that increase patient survival and overall survival. Currently, tumor immunotherapy is considered as the fourth mainstay of cancer therapy (in parallel with surgery, radiotherapy and chemotherapy), and the therapeutic effect of combined immunotherapy on part of malignant tumors on immunocompetent patients is unprecedented and has become a first-line therapeutic approach.

Current immunotherapy is mainly: t cell checkpoint inhibitors (e.g., PD-1 mab therapy, adoptive T cell therapy, chimeric antigen receptor T cell therapy (CAR-T), T cell receptor chimeric T cell (TCR-T) therapy) have achieved significant therapeutic effects in clinical therapies. However, these immunotherapies all have certain limitations. Although CAR-T cell therapy has better efficacy in hematological tumors, its toxic side effects are also evident, the most significant of which is the induction of Cytokine Release Syndrome (CRS). In addition, autoimmune diseases may also be caused. PD-1 mAbs have only a response rate of less than 30% in clinical treatment. Based on the limitations of current cancer immunotherapy, there is an urgent clinical need to develop new cancer immunotherapy and platform.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide an application of a NIH3T3 mouse fibroblast autophagy body in preparing a vaccine for treating melanoma, and the invention can activate an immune system by genetically engineering the mouse fibroblast NIH3T3 autophagy body to simultaneously wrap new antigens of melanoma and MAP1LC3, thereby achieving the purpose of inhibiting the growth of the melanoma.

In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:

the invention provides an application of an NIH3T3 mouse fibroblast autophagosome in preparing a vaccine for treating melanoma, wherein the NIH3T3 mouse fibroblast autophagosome is prepared from NIH3T3 mouse fibroblasts stably expressing a new melanoma antigen and MAP1LC 3.

Preferably, the NIH3T3 mouse fibroblasts stably expressing the melanoma tumor neoantigen and MAP1LC3 are prepared by infecting NIH3T3 mouse fibroblasts with lentiviruses expressing the melanoma tumor neoantigen and MAP1LC3, respectively.

Preferably, the NIH3T3 mouse fibroblast autophagosome is prepared by:

inducing cell autophagy of NIH3T3 mouse fibroblast which stably expresses melanin tumor neoantigen and MAP1LC3, and extracting to obtain the small body of the NIH3T3 mouse fibroblast autophagy.

In a second aspect the invention provides a vaccine for the treatment of melanoma, the vaccine comprising NIH3T3 mouse fibroblast autophagosomes.

Preferably, the vaccine further comprises an adjuvant.

Preferably, the adjuvant is GM-CSF and/or HMGB1.

The third aspect of the present invention provides a method for preparing the vaccine for treating melanoma, comprising the steps of:

(a) Dissolving an adjuvant in double distilled water and precooling to obtain an adjuvant solution;

(b) Adding the mixed solution of hyaluronic acid and Pluronic F-127 into an adjuvant solution, and uniformly mixing to obtain liquid hydrogel;

(c) Under ice bath condition, adding the NIH3T3 mouse fibroblast autophagosome into the liquid hydrogel to obtain the vaccine for treating melanoma.

Preferably, when the adjuvant is GM-CSF and/or HMGB1, the concentration of GM-CSF in the adjuvant solution is 15-25 ng/mL and the concentration of HMGB1 is 0.8-1.2 mug/mL.

Preferably, the addition amount of the hyaluronic acid is 1.5-2.5% of the mass of the adjuvant solution; the addition amount of Pluronic F-127 is 20-30% of the mass of the adjuvant solution.

Preferably, the concentration of the NIH3T3 mouse fibroblast autophagosome is 2-3 mg/mL.

Compared with the prior art, the invention has the beneficial effects that at least:

according to the invention, the mouse fibroblast NIH3T3 autophagosome is modified through genetic engineering, so that the mouse fibroblast NIH3T3 autophagosome is coated with a new melanoma antigen and MAP1LC3 at the same time, and the immune system can be activated, thereby achieving the purpose of inhibiting the growth of melanoma; in addition, the method adopts two medicaments to jointly treat NIH3T3 cells, and can rapidly extract a large amount of autophagosomes by a grinding and centrifuging method.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. Like elements or portions are generally identified by like reference numerals throughout the several figures. In the drawings, elements or portions thereof are not necessarily drawn to scale.

FIG. 1 is a laser confocal plot of NIH3T3 mouse fibroblast cell lines stably expressing MAP1LC3 and Neoantigins in example 1 of the present invention;

FIG. 2 shows the increase in GFP-LC3 expression level after autophagy induced by NIH3T3 cells in example 1 of the present invention;

FIG. 3 is a transmission electron microscope image of the mouse fibroblast autophagosome of NIH3T3 in example 2 according to the invention;

FIG. 4 is a laser confocal plot of mouse DC cells versus NIH3T3 mouse fibroblast autophagy mice uptake in example 4 of the present invention;

FIG. 5 is a flow chart showing in vitro activation of DC cells by melanoma vaccine according to example 5 of the present invention;

FIG. 6 is a flow chart showing changes in DC cell maturation in lymph nodes of mice following administration of melanoma vaccine according to example 6 of the present invention;

FIG. 7 is a photograph of in vivo images of mice in example 7 of the present invention in a PBS group and in a LC3/Mut treated group of mice in a post-melanoma postoperative model;

FIG. 8 is a graph showing tumor volume change and survival rate of mice in PBS group and LC3/Mut treatment group of mouse melanoma postoperative model in example 7 of the present invention;

FIG. 9 is a horizontal flow chart of tumor-infiltrating T cell immune responses of the PBS group and the LC3/Mut treatment group of the mouse melanoma postoperative model in example 7 of the present invention;

FIG. 10 is a photograph of in vivo images of mice in example 8 of the present invention in a PBS group and in a LC3/Mut treated group of mice in a melanoma lung metastasis model;

FIG. 11 is a photograph of lung and HE staining pattern of lung tissue of mice in PBS group and LC3/Mut treated group of the lung metastasis model of mouse in example 8 of the present invention.

FIG. 12 is a graph showing survival rates of PBS group and LC3/Mut treated group of mice melanoma lung metastasis model in example 8 of the present invention.

Detailed Description

Embodiments of the technical scheme of the present invention will be described in detail below with reference to the embodiments. The following examples are only for more clearly illustrating the technical aspects of the present invention, and thus are merely examples, and are not intended to limit the scope of the present invention.

It is noted that unless otherwise indicated, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

Example 1

The present example is a method for establishing NIH3T3 mouse fibroblasts stably expressing a melanoma tumor neoantigen and MAP1LC 3:

the gene fragments for expressing mouse Neoantigen and MAP1LC3 were cloned into pLenti-C-mCherry and pLenti-EGFP-N lentiviral expression vectors to construct pLenti-Neoantigen-mCherry and pLenti-EGFP-MAP1LC3 lentiviral expression plasmids (both purchased from Beijing-England Biotech Co., ltd., and packaging plasmid). The correct construction of neoantigen and MAP1LC3 expression vectors was confirmed by Gene sequencing (served by the Optimago of the NCBI website Gene database;

amplification of plasmids and packaging purification of viruses:

transferring the TransStbl3 competent cells from a refrigerator at the temperature of minus 80 ℃ to ice for thawing, adding 500ng of plasmid into the competent cells on an ultra-clean workbench, gently mixing the competent cells and standing for 30min; heating in a water bath at 42deg.C for 45s, and placing on ice for 5min; adding 500 mu L of LB culture medium on an ultra-clean workbench, placing the culture medium on a shaking table at 37 ℃ and shaking the culture medium at 220rpm for 1.5 hours; transferring to a 10mL centrifuge tube in an ultra-clean workbench, adding 9mL of LB culture medium containing 60 mug/mL ampicillin, placing in a shaking table at 37 ℃ and shaking at 220rpm for 12 hours; extracting plasmids according to the operation steps of a high-purity plasmid small extraction kit (Tiangen brand), detecting the concentration and freezing; HEK 293T cells are passaged to a 10cm culture dish at 50% density, and the density reaches about 80% after the culture in an incubator is attached; 2 centrifuge tubes were prepared in a biosafety cabinet, 1mL MEM medium was added, one tube was added with the plasmid of interest and the packaging plasmid, the plasmid of interest was 6. Mu.g, the packaging plasmid pH1 was 4.5. Mu.g, and pH2 was 1.5. Mu.g; another tube was added 12. Mu.L Lipofectamine 2000 transfection reagent; after standing for 5min, the two tubes were mixed and left standing for 30min. The HEK 293T cell culture dish is taken out, the culture medium is sucked out, 4mL of MEM culture medium is added, then the mixed solution of plasmid and transfection reagent is added, the mixture is gently mixed, the mixture is placed in an incubator for 8 hours, and then the DMEM culture medium containing 10% fetal calf serum and 1% penicillin/streptomycin is replaced for continuous culture. Collecting the supernatant containing the lentivirus at 24h,48h and 72h respectively, and replacing the fresh culture medium; all collected virus solutions were centrifuged at 500g for 10min at 4℃and the supernatant was collected in a biosafety cabinet and filtered through a 0.45 μm filter according to 4:1 adding lentivirus concentrated solution in proportion, and placing the solution in a refrigerator at 4 ℃ for overnight; centrifuging the mixed solution at 4 ℃ for 25min at 3500g, discarding the supernatant at 4 ℃ for 5min at 3500g, and sucking the residual liquid by a pipette; adding PBS with 1% of the original volume for resuspension, sub-packaging and freezing.

Cell lines stably expressing neoanti and MAP1LC3 were obtained using lentiviral infection:

NIH3T3 cells were passaged at 50% density into six well plates, placed in culture until fully adherent and grown stably; the old culture medium is sucked off in a biosafety cabinet, 500 mu L of fresh culture medium is added to each hole, polybrene is added to make the final concentration 8 mu g/mL; adding one part of EGFP-MAP1LC3 virus into each hole, uniformly mixing, and then placing the mixture into an incubator for continuous culture; after 72h, observing under a fluorescence microscope, and judging whether the MAP1LC3 fused with the green fluorescent protein (EGFP) is successfully expressed or not through fluorescence intensity; the cells in each hole are subcultured to a new six-hole plate, puromycin with different concentrations is added for drug resistance screening, the expression condition of the cells is judged through fluorescence intensity, and after several generations of screening, the NIH3T3 cell strain capable of stably expressing EGFP-MAP1LC3 is finally obtained; and then, infecting a cell strain capable of stably expressing MAP1LC3 with a virus of which red fluorescent protein (mCherry) is fused with Neoantigins, adding hygromycin B with different concentrations for screening, judging the expression condition of the cell Neoantigins through fluorescence intensity, and finally obtaining the cell strain capable of simultaneously stably expressing EGFP-MAP1LC3 and Neoantigins-mCherry after repeated passage screening.

Stable expression of cell lines MAP1LC3 and Neoantigens was verified by laser confocal microscopy:

the screened cell line is passaged to a confocal laser culture dish at 50% density, cultured overnight, and after the cells are completely adhered and have good growth state, the culture medium is sucked and PBS is added for cleaning; the PBS was removed and 4% paraformaldehyde was added and the mixture was allowed to stand at room temperature for 15min; after PBS cleaning, 1mL of DAPI solution with the concentration of 1 mug/mL is added, and the mixture is incubated for 10min in a dark place; PBS is washed for 3 times, each time for 5min; the PBS was blotted off, 20. Mu.L of an anti-fluorescence quencher was added, and the result was observed under a laser confocal microscope as shown in FIG. 1,

further, protein immunoblotting (Western Blot) was used to detect the expression of EGFP-MAP1LC3 protein. The screened cells stably expressing MAP1LC3 were lysed for preparation and separated by 10% polyacrylamide gel electrophoresis. Electrotransfer of proteins on gel to PVDS membrane, sealing with skimmed milk powder for 1h, incubating GFP primary antibody at 4deg.C overnight; incubating the secondary antibody for 1h at room temperature, and finally developing, detecting GFP-MAP1LC3 protein signals, wherein the detection result is shown in figure 2;

as can be seen from fig. 1 and 2: a large amount of green fluorescent dot-like aggregation can be observed under a laser confocal microscope, which proves that LC3 is aggregated on an autophagosome membrane, a large amount of autophagosomes are formed, and the neoanti (Mut-mCherry) can be successfully wrapped in the autophagosome according to the co-localization with the autophagosome EGFP-LC 3. After the NIH3T3 cells are induced to autophagy, the expression level of GFP-LC3 is obviously increased through Western Blot experiments. Thus, stable expression of MAP1LC3 and Neoantigens in NIH3T3 cells was verified.

Example 2

This example is a method for preparing an NIH3T3 mouse fibroblast autophagosome comprising the steps of:

amplifying NIH3T3 stably expressing MAP1LC3 and Neoantigins into 15cm dishes; when the cell density reaches more than 80%, changing fresh culture medium, adding rapamycin with the final concentration of 1 mu mol/L and chloroquine with the final concentration of 30 mu mol/L, continuously culturing for 24 hours, sucking the culture medium, washing twice with PBS, adding 5mL of PBS, and gently scraping and collecting cells by using a cell scraper; centrifuging the collected cells at 4 ℃ and 800rpm for 5min, collecting cell sediment, adding 3mL PBS containing protease inhibition to resuspend the cells, placing a glass homogenizer on ice, adding cell suspension, homogenizing for 30-40 times in ice bath, and collecting homogenate; centrifuging the homogenate at 4deg.C for 5min and 1000g for 5min, collecting supernatant, centrifuging again at 4deg.C for 5min and 1000g for 30min, collecting supernatant, collecting precipitate, adding 500 μl of protease inhibitor-containing PBS, and resuspending to obtain separated autophagosome;

an ice box filled with ice is prepared, an electron microscope carrier net is clamped by forceps, and the forceps are fixed by a clamp to be closed to prevent falling. The forceps were fixed above the ice box using an adhesive tape, 10 μl of autophagosome suspension was dropped on the support film surface of the carrier net, and after standing for 5min, the remaining liquid was sucked off the edge using filter paper, and the drop was repeated six times. 10 μl of 3% uranium acetate was added dropwise, and the mixture was left to stand for 5min for staining, and the liquid was sucked from the edge with filter paper. Airing the carrier net at room temperature, observing the autophagy body by using a 120kV transmission electron microscope, and photographing, wherein the photographing result is shown in figure 3;

as can be seen from fig. 3: autophagosomes with a bilayer membrane structure were successfully isolated by extraction.

Example 3

The embodiment is a preparation method of a vaccine for treating melanoma, comprising the following steps:

to ddH ₂ Adding GM-CSF and HMGB1 into O to make the final concentration be 20ng/mL and 1 mug/mL, precooling;

hyaluronic acid (HA, 2 wt%) and Pluronic F-127 (25 wt%) were mixed, added to the pre-chilled solution and mixed well, and the mixture was placed in a refrigerator at 4℃overnight to allow the materials therein to dissolve completely and form a homogeneous liquid hydrogel;

a corresponding volume of hydrogel was placed on ice, and the autophagosome prepared in example 2 was added to the hydrogel at a final concentration of 2.5mg/mL, mixed well, and kept in an ice bath to obtain a vaccine for treating melanoma (designated LC3/Mut or Mut/LC 3).

Example 4

This example is the in vitro culture of mouse bone marrow derived dendritic cells (BMDCs) and uptake of autophagosomes:

all femur and tibia were removed and periosseous musculature was removed with scissors at the sacrifice of 6-8 week old C57BL/6 mice. The bones were moved into an ultra clean bench, soaked in 75% alcohol for 5min for sterilization and sterilized, and washed 2 times with sterile PBS. The two ends of the bone are cut off, and the syringe needle containing PBS is inserted into the bone marrow cavity to repeatedly wash out the bone marrow. Transferring the bone marrow suspension into a centrifuge tube, and filtering small fragments and muscle tissues by using a 200-mesh nylon net; centrifuging at 1200rpm for 5 minutes, and discarding the supernatant; the cells were resuspended with erythrocyte lysate and incubated for 5 minutes at room temperature. The mixture was centrifuged at 1200rpm for 5 minutes, and the red supernatant was discarded. The resulting cells were then cultured in 1640 complete medium containing 10% FBS (containing 20ng/ml GM-CSF and 10ng/ml IL-4), and the resulting bone marrow cells were subjected to cell counting to adjust the concentration to 1X10 ⁶ Per ml, 1ml per well, added to a 12-well plate; thereafter, 3/4 of the volume of fresh culture medium was replaced every 2 days. On day 6, the culture solution is gently blown, and the suspension cells and loose cells growing on the wall are collected;centrifuging at 1200rpm for 5 minutes, and discarding the supernatant; cells were resuspended and counted using the above-mentioned media and the concentration was readjusted to 1x10 ⁶ Adding/ml into 24-well plate, and culturing for 1-2 days;

adding isolated and purified autophagosomes into a BMDC cell culture medium for in-vitro culture and differentiation, incubating for 24 hours in an incubator, collecting cell suspension, transferring into a centrifuge tube, centrifuging for 5min at 1200rmp, discarding supernatant, resuspending cell sediment with PBS, adding into a laser confocal culture dish, adding Hoechst dye liquor for dying nuclei, and observing the ingestion condition of BMDC cells on autophagosomes under a laser confocal microscope, wherein the result is shown in figure 4;

as can be seen from fig. 4: autophagosomes (LC 3/Mut-Autophagosomes) containing the neoantigen were successfully taken up by BMDC cells.

Example 5

This example is a study of the in vitro activation of dendritic cells by the melanoma vaccine of example 3:

500. Mu.L each of PBS group and LC3/Mut group preparations was placed in a Transwell chamber of 8 μm and allowed to solidify, and then the cells were placed in a 24-well plate in which dendritic cells were cultured, and after two days of culture in an incubator at 37℃the DCs were collected for flow assay. After collection of the cell suspension, centrifugation was performed at 1200rpm for 5 minutes, the supernatant was discarded, resuspended in PBS and repeated 3 times. Re-suspending with a stabilizing buffer to obtain 1×10 ⁵ The anti-CD11c-APC, anti-CD80-FITC, anti-CD86-PE and anti-MHC II-Percp/Cy5.5 antibody were used in a volume ratio of 1:100 DC cells of each group were stained in ice for 30 minutes in the absence of light, centrifuged at 1200rpm for 5 minutes, resuspended in stabilizing buffer, and assayed 3 times with a flow analyser, the results of which are shown in FIG. 5;

as can be seen from fig. 5: the proportion of cells highly expressing CD80/86, CD40 and MHC II in the DC cells of the LC3/Mut group is obviously increased, which indicates that the maturation proportion is obviously increased compared with that of the PBS group, and the LC3/Mut has strong activation effect on the DC cells.

Example 6

This example is the melanoma vaccine in vivo activation assay of example 3:

after the mice are randomly grouped, different experimental preparations are injected into the back of the mice to be subcutaneously, as followsThe following groups are used for experiments: (1) PBS, (2) vaccine preparation (LC 3/Mut) containing LC 3/Mut-Autophagosomes. 24h after injection treatment, mice were sacrificed, the collected mice lymph nodes were washed in PBS and placed in EP tubes, PBS containing 2% FBS was added, and minced with scissors as much as possible. The fragments were gently ground on a 70 μm cell screen and continuously rinsed with PBS containing 2% FBS using a pipette, and collected to give a single cell suspension. After centrifugation, the cells were resuspended and counted using a stabilizing buffer, aliquoted into 1.5mL EP tubes and grouped, and adjusted to a cell suspension volume of 50. Mu.L and a cell number of 1X10 per tube ⁵ . anti-CD11c-APC, anti-CD80-FITC, anti-CD86-PE, anti-CD40-PE and anti-MHC II-Percp/Cy5.5 stream antibody were purified according to a 1:100 volume ratio was added to each group of cell tubes and incubated on ice for 30min for staining in the dark. Centrifuging at 1200rpm for 5min, adding stabilizing buffer, re-centrifuging, cleaning, repeating for 3 times, and detecting with flow cytometry, wherein the result is shown in FIG. 6;

as can be seen from fig. 6: vaccine injected (2) in LC3/Mut mice lymph node CD11c compared to (1) PBS group ⁺ CD80 in DC cells of (C) ⁺ CD86 ⁺ Cell, CD40 ⁺ Cell and MHC II ⁺ The proportion of cells is obviously increased, which indicates that the maturity of DC cells in lymph nodes is higher, and indicates that neoantigen has strong activating effect on immune system. The prepared autophagosome tumor vaccine can effectively recruit DC cells in a body, promote uptake, differentiation and maturation of the DC cells and migrate to lymph node presenting antigens to start an immune system.

Example 7

The embodiment is a construction and treatment experiment of a mouse melanoma postoperative model:

will be 1X10 ⁶ The B16F10-Luciferase cells were inoculated subcutaneously into C57BL/6 mice aged 6-8 weeks, and after one week, the tumors were allowed to grow to about 100mm ³ Size, mice were anesthetized with isoflurane and surgically resected with about 1% tumor tissue left to simulate residual tumor tissue that was not completely or hardly cleared in clinical surgery, after which the incision was closed with a mouse wound clip.

After the next post-operative day mice were recovered, the mice were randomized into 2 groups of 10 mice each, each group being given a different therapeutic treatment. The tumor vaccine experimental group is used for subcutaneously inoculating the preparation to the back of a mouse, the dosage of autophagosome in gel vaccine is 25mg/kg, the injection is carried out 1 st time after operation, the injection is carried out 2 nd time after operation, and the total injection is carried out 2 times; control group was injected with 200 μl of PBS tail vein. Tumor growth was recorded periodically by a small animal in vivo imaging system, mice were injected with fluorescein substrate intraperitoneally, after 10min, mice were anesthetized with isoflurane, transferred into a small animal in vivo imager for detection of bioluminescence and imaging observations, and the results are shown in fig. 7. The tumor volume of the mice is measured and recorded regularly, the formula is long diameter multiplied by short diameter 2/2, the weight of the mice is weighed and recorded regularly, the health condition is observed, 5 days after the administration is finished, the last living imaging is carried out, part of the mice are dead, recurrent tumor tissues are taken for analysis, the rest of the mice are continuously fed, the survival number is recorded, and the survival rate is counted, and the result is shown in figure 8.

Mice were sacrificed using cervical dislocation, intact mouse tumor tissue was sheared and weighed. After washing with PBS, a portion of the tissue was cut out and placed in an EP tube for flow analysis experiments. PBS containing 2% FBS was added to the EP tube and the tumor tissue was minced as much as possible with scissors. The fragments were gently ground on a 70 μm cell screen and continuously rinsed with PBS containing 2% FBS using a pipette, and collected to give a single cell suspension. The tumor cell suspension is divided into two parts, wherein one part is used for detecting and analyzing the cell surface molecules, and the other part is used for detecting and analyzing the cell surface molecules and intracellular cytokines. Single cell suspensions for cell surface molecular detection were centrifuged and resuspended and counted using a starting buffer, dispensed into 1.5mL EP tubes and grouped, and adjusted to a cell suspension volume of 50. Mu.L and a cell number of 1X10 per tube ⁵ . And adding the flow antibody for detecting the cell surface molecules such as CD3, CD4, CD8 and the like into each group of cell tubes according to the recommended proportion, and incubating for 30min on ice in a dark place for dyeing. Centrifugation at 1200rpm for 5min, suspension with stirring buffer was added, and the washing was repeated 3 times by centrifugation again, and detection was performed by flow cytometry. Single cell suspensions for cell surface molecule and intracellular cytokine detection were centrifuged to harvest cells, and grown in 1640 medium with 50ng/mL PMA and 1. Mu.g/mL ionomycinCulturing in culture solution, culturing in incubator at 37deg.C for 1 hr, adding brefeldin A to final concentration of 1-50 μm, and culturing for 3-6 hr. The cell suspension was collected, centrifuged, the supernatant was discarded, and the supernatant was resuspended in PBS, and the washing was repeated 2 times by centrifugation again. Resuspended and counted using a stabilizing buffer, aliquoted into 1.5mL EP tubes and grouped, and adjusted to a cell suspension volume of 50. Mu.L per tube and a cell number of 1X10 ⁵ . Flow antibody staining for detection of cell surface molecules was performed as in the previous step. After that, the mixture was centrifuged and washed 2 times with PBS, and then 4% paraformaldehyde was added for fixation, followed by incubation at room temperature for 20min in the dark. After centrifugation, the supernatant was discarded, resuspended in PBS, and washed again by centrifugation, and repeated 2 times. Cell membranes were perforated by resuspension using a starting buffer containing 0.2% Trixton-x100, incubated for 20min at room temperature. Cells were collected by centrifugation and resuspended in 50. Mu.L per tube using a starting buffer containing 0.01% Trixton-x 100. Adding a flow antibody for detecting intracellular cytokines according to the recommended proportion, and incubating for 30min on ice in a dark place for dyeing. After completion, the cells were collected by centrifugation, washed twice with a stationary buffer, detected by a flow cytometer, and analyzed by FlowJo, and the results are shown in fig. 9.

As can be seen from fig. 7 to 9: on day 7, all mouse tumor sites have obvious fluorescence, and the fluorescence intensity components are randomly distributed, but most of the fluorescence intensity components are not greatly different, so that the success of the tumor inoculation is shown; tumor resection operation is carried out on the 7 th day, administration is started on the 8 th day, and observation is carried out on the 9 th day of the experimental flow, so that the fluorescence of the tumor part of the mouse is weak, but still the tumor part of the mouse is visible, and the model establishment of residual and recurrent postoperative is successful; on day 13, the fluorescence of the tumor sites of each group of mice started to be different, but the overall fluorescence intensity was not high, probably because the growth of recurrent tumors did not reach a rapid growth stage temporarily, and the fluorescence intensity of the PBS group increased relatively more; on day 17, the fluorescence intensity of all mice tumor sites in the PBS group was very high and increased faster than the last imaging, and the LC3/Mut group overall increased more slowly. The LC3/Mut tumor vaccine can effectively slow down the growth rate of tumors, thereby prolonging the survival time and improving the survival rate. CD4 and CD8 are different T cell surface molecules and can be combined with MHC II and MHC I respectively, and CD8 ⁺ T cells are typically cytotoxic T Cells (CTL), CD4 ⁺ T is a helper T cell, and both of these activated T cells are the main contributors to adaptive immunity. Activated CD8 ⁺ T、CD4 ⁺ T cells can also secrete cytokines such as IFN-gamma, TNF-alpha and the like to enhance the immune system effect and indirectly kill tumors. CD8 in tumor tissue of LC3/Mut group combination treated mice ⁺ T、CD4 ⁺ The secretion of IFN-gamma and TNF-alpha by T cells is obviously increased, and the autophagosome tumor vaccine can effectively activate the specific anti-tumor immune response of the organism, so that the infiltration of activated T cells at the tumor part is increased, and the specific anti-tumor function is maintained.

Example 8

The embodiment is a construction and treatment experiment of a mouse melanoma lung metastasis model

Will be 2X 10 ⁵ The B16F10-Luciferase cells were injected into C57BL/6 mice aged 6-8 weeks by tail vein to simulate clinical blood metastasis of melanoma and form a lung metastasis model. Tumor vaccine was inoculated 1 st day after molding and 2 nd day after 7 days. The mice were weighed and recorded periodically and observed for health. Mice were periodically injected intraperitoneally with a fluorescein substrate and the growth of lung tumors was observed using a small animal in vivo imaging system, the results of which are shown in fig. 10. At day 20 of the start of dosing, the last in vivo imaging was performed. After that, a part of the mice was sacrificed, and after dissection, the lungs were taken out for observation and photographing, and a part of the lung tissues was taken for HE-stained photographing (served by wuneseir biotechnology limited) and the results are shown in fig. 11. The number of surviving mice was recorded and the survival rate was counted, and the results are shown in fig. 12;

as can be seen from fig. 10 to 12: on day 5 of tumor development, the lungs of most mice begin to appear weakly fluorescent, demonstrating that melanoma begins to metastasize to and grow in the lungs; on day 10, the lung fluorescence of the PBS group mice rapidly increased, indicating that tumor growth had entered log phase, and that the lung tumor growth was retarded in the LC3/Mut group and the aPD-1 (G5) group mice; on day 15, the tumor fluorescence of the PBS group mice is extremely strong, the lung fluorescence of the LC3/Mut group mice is not obviously enhanced, and the tumor growth is slow; on day 20, the lung tumor fluorescence of the PBS group mice was also enhanced, and the lung fluorescence of the LC3/Mut group mice increased at a slower rate. After the end of the dosing, part of the mice were sacrificed, the whole mouse lung tissue was removed, observed and sectioned for HE staining. The results show that the PBS group mice had their lungs spread over the tumor, the LC3/Mut mice had a smaller number of lung lesions. The LC3/Mut tumor vaccine can effectively slow down the growth rate of lung metastasis tumor, thereby prolonging the survival time and improving the survival rate.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention, and are intended to be included within the scope of the appended claims and description.

Claims (10)

1. The use of an NIH3T3 mouse fibroblast autophagy minibody in the preparation of a vaccine for the treatment of melanoma, wherein the NIH3T3 mouse fibroblast autophagy minibody is prepared from NIH3T3 mouse fibroblasts stably expressing a melanoma tumor neoantigen and MAP1LC 3.
2. 2. The use according to claim 1, wherein the NIH3T3 mouse fibroblasts stably expressing the melanoma tumor neoantigen and MAP1LC3 are prepared by infecting NIH3T3 mouse fibroblasts with lentiviruses expressing the melanoma tumor neoantigen and MAP1LC3, respectively.
3. 3. The use according to claim 1, wherein the NIH3T3 mouse fibroblast autophagosome is prepared by:

   inducing cell autophagy of NIH3T3 mouse fibroblast which stably expresses melanin tumor neoantigen and MAP1LC3, and extracting to obtain the small body of the NIH3T3 mouse fibroblast autophagy.
4. 4. A vaccine for the treatment of melanoma, comprising NIH3T3 mouse fibroblast autophagosomes.
5. 5. The vaccine of claim 4, further comprising an adjuvant.
6. 6. The vaccine of claim 5, wherein the adjuvant is GM-CSF and/or HMGB1.
7. 7. A method of preparing a vaccine for the treatment of melanoma according to any one of claims 4 to 6, characterized in that it comprises the steps of:

   (a) Dissolving an adjuvant in double distilled water and precooling to obtain an adjuvant solution;

   (b) Adding the mixed solution of hyaluronic acid and Pluronic F-127 into an adjuvant solution, and uniformly mixing to obtain liquid hydrogel;

   (c) Under ice bath condition, adding the NIH3T3 mouse fibroblast autophagosome into the liquid hydrogel to obtain the vaccine for treating melanoma.
8. 8. The method according to claim 7, wherein when the adjuvant is GM-CSF and/or HMGB1, the concentration of GM-CSF in the adjuvant solution is 15-25 ng/mL and the concentration of HMGB1 is 0.8-1.2. Mu.g/mL.
9. 9. The preparation method of claim 7, wherein the addition amount of the hyaluronic acid is 1.5-2.5% of the mass of the adjuvant solution; the addition amount of Pluronic F-127 is 20-30% of the mass of the adjuvant solution.
10. 10. The method according to claim 7, wherein the concentration of the NIH3T3 mouse fibroblast autophagosome is 2 to 3mg/mL.